The present invention relates to materials and methods for transferring nucleic acid encoding a polypeptide for treating a disease or disorder into populations of quiescent cells such as haematopoietic stem cells (HSCs), using retroviral packaging cell lines and retroviral particles expressing and displaying a growth factor such as stem cell factor (SCF) on the cell surface or as a fusion with a viral envelope protein. The present invention also relates compositions comprising the retroviral packaging cell lines and retroviral particles, and their use in methods of medical treatment, in vivo and ex vivo.
The development of somatic gene therapy as a treatment for single gene inherited diseases and some acquired conditions, such as certain types of cancer, represents one of the most important technical advances in medicine. Blood related disorders such as the X-linked immunodeficiencies, or chronic granulomatous disease (CGD), are amongst the most favourable candidates as model systems for the evolution of this technology. The general feasibility of gene therapy for disorders of this type has been amply demonstrated by the results obtained in the treatment adenosine deaminase dependent severe combined immunodeficiency (ADA-SCID) using peripheral blood T-cells.
However, many problems stand in the way of the realisation of the promise of these techniques. For example, in the experiments described above, the T-cells including the genes required by the patients are not immortal, requiring the therapy to be repeated at regular intervals. Further, attempts to effect a permanent correction, for example by gene transfer into pluripotent haematopoietic stem cells (PHSC), have thus far been unsuccessful.
There are a number reasons for this. Firstly, PHSC are very rare in the bone marrow cell population, and so although work has been done on bone marrow cell culture, it is very difficult to draw conclusions from this work regarding PHSCs. Further, in humans there is a dearth of markers to identify PHSC and, at present, the most reliable marker of immature human bone marrow cells is the CD34 antigen, which marks about 1-2% of total marrow cells. However, probably only about 0.1% of these CD34+ cells are true PHSC. In addition, there are no wholly reliable assays for human PHSC, unlike murine systems, where the rescue of lethally irradiated individuals can be used to test for PHSC.
Recently, a method to enrich for PHSC has been described by Beradi et al (Science, 267, 104-108, (1995)) which exploits the quiescence of PHSCs as a basis for their functional isolation. In this method, bone marrow cells were incubated for 7 days in the presence of the cytokines stem cell factor (SCF) and IL-3, to stimulate division in all of the progenitor cells, but not in true PHSC. The cytotoxic agent, 5-fluorouracil (5-FU), was then added to these cultures, resulting in the death of all dividing cells in the culture. However, quiescent cells, including PHSC which average only 1 in 105 of the original cells, were spared in this process. Accordingly, the authors reported obtaining an enriched population of cells having the characteristics of true PHSC.
However, the authors of this paper were unable to find any combination of cytokines that was able to stimulate these cells to divide, other than incubation in long term marrow culture (LTC), which also leads to their differentiation.
Thus, although, this method produces highly enriched populations of PHSC, it is their quiescence, the very property exploited for their isolation by Beradi et al, that still represents the most significant hurdle limiting current gene therapy protocols. This is because most highly developed vector systems presently used for gene transduction are based on murine retroviruses and these viruses (and the vectors derived from them) are unable to stably integrate their genome into non-dividing cells, rendering PHSCs refractory to retroviral gene transfer.
Previously, we presented an abstract at the European Working Group for Gene Therapy in November 1994 disclosing that a retroviral cell line containing a viral vector incorporating nucleic acid encoding GCD and expressing stem cell factor on its surface was able to achieve improved rates of transduction in a bone marrow cell culture. However, as mentioned above this cell culture contains a very low proportion of PHSC, and this treatment would not be expected to stimulate the PHSC to divide or to allow the stable integration of the nucleic acid encoding GCD into the PHSC genome. An important fact underlying this expectation is that in Beradi et al, stem cell factor was one of the cytokines used to stimulate selectively division in the most of the cells in marrow cell culture (but not the PHSC), allowing them to be killed to leave the enriched population of stem cells.
The present invention is based on the unexpected finding that it is possible to get haematopoeitic stem cells to cycle transiently during the period of exposure to vectors incorporating nucleic acid encoding a desired protein or polypeptide by exposing them to bound growth factors such as stem cell factor. This observation means that contrary to prior expectations, a population of quiescent cells such as PHSC can be used as targets for vectors incorporating nucleic acid encoding a desired protein or polypeptide, provided that the cells are additionally exposed to a surface bound growth factor, e.g. stem cell factor expressed by a retroviral packaging cell line so that it is bound on the cell surface or expressed as a fusion with an envelope protein of retrovirus so that the growth factor is displayed on the surface of the retrovirus.
Without wishing to be bound by any particular theory, we believe that the exposure of the quiescent cells to the membrane or surface bound growth factor induces them to start dividing, so that the nucleic acid, e.g. packaged in retroviral particles produced by a retroviral packaging cell line, can infect the cells and become incorporated into their genomes which become accessible during cellular division when the nuclear membrane dissolves. This method has the advantage that it can be adapted for the treatment of a wide variety of disorders, by incorporating nucleic acid encoding an appropriate protein or polypeptide into the vector. A further advantage of the method is that by stimulating the quiescent cells to differentiate at the time of gene transfer, preferential amplification of the transduced cells relative to the non-transduced cells can be achieved.
Accordingly, in a first aspect, the present invention provides a retroviral packaging cell line transformed with a viral vector comprising nucleic acid encoding a polypeptide for treating a disease or disorder, the retroviral packaging cell line being capable of expressing nucleic acid encoding a growth factor so that the growth factor is (i) displayed on the cell surface or (ii) expressed as a fusion with a viral envelope protein so that the growth factor is displayed on the surface of viral particles,
wherein the cell line packages the nucleic acid encoding the polypeptide in viral particles produced by the retroviral packaging cell line, the cell line being for use in a method of medical treatment of a disease or disorder that responds to the polypeptide.
In this aspect, the retroviral packaging cell line includes nucleic acid encoding viral envelope protein so that the cell line can produce viral particles and package the nucleic acid encoding the polypeptide for treating the disease or disorder in them.
In this application, xe2x80x9cquiescentxe2x80x9d refers to cells that are unlikely to enter mitosis within the next 24 hours in the absence of appropriate growth stimulus. Preferably, the population of quiescent cells are enriched in haematopoeitic stem cells, for instance by employing the isolation method of Beradi et al (supra) using bone marrow cells. Other quiescent cell types suitable for use in the invention include resting T-lymphocytes, B-lymphocytes and monocytes, stem cells of non-haematopoietic tissues such as liver and muscle, epithelial stem cells in skin, gut, bladder and airways, vascular endothelial cells, quiescent neoplastic cells and germ cells such as sperm progenitors.
In a further aspect, the present invention provides retroviral particles displaying surface bound growth factor as a fusion with an envelope protein, the particles being produced by the retroviral packaging cell line as set out above.
In one embodiment, the surface bound growth factor is provided by engineering the retroviral packaging cell line to express growth factor on its surface by transfecting the cell line with nucleic acid encoding the growth factor.
In an alternative embodiment, a retroviral vector expressing surface bound growth factor (e.g. SCF) could be prepared by constructing a packaging cell line engineered to produce a chimeric retroviral envelope protein fused to all or part of the growth factor. The growth factor can be used to replace the natural binding domain of the envelope protein, or can be fused directly to the C- or N- terminus of a retroviral envelope protein. Such chimeric envelopes have been described for use in retroviral targeting (7-9). In this embodiment, the retroviral packaging cell line may also display the growth factor-envelope protein fusion on the surface of the retroviral packaging cell line. The chimeric envelope could be expressed as the sole viral envelope protein in an attempt to target the retrovirus to stem cells, as well as to transduce a growth signal, or in concert with the xe2x80x9cwild typexe2x80x9d envelope protein, to induce growth in growth factor responsive target cells, without targeting to a specific cell type. The former strategy is more applicable to an in vivo situation, the latter to an in vitro transduction process. An example of this is the expression of the growth factor as a fusion with viral envelope SU protein of murine leukemia virus (MLV).
In some instances, expressing the growth factor as a fusion with a viral envelope protein, may lead to the nucleic acid encoding the polypeptide not being incorporated into the genome of the target quiescent cells. This can be overcome by introducing a cleavable linker between the viral envelope protein and the growth factor so that the growth factor can be cleaved from the viral particle by addition of a cleaving agent, typically once the quiescent cells start dividing. An example of such a system is the use of a chimeric envelope protein in which viral envelope protein is linked to a factor Xa linker which is in turn linked to the growth factor. In this system, factor Xa protease can be used to cleave the growth factor from the surface of the viral particles, so that the particles can transfer the nucleic acid encoding the polypeptide to the target cells where it can be incorporated into their genomes.
Preferably, the surface bound growth factor is FLT3-ligand, or stem cell factor, also known as mast cell growth factor, kit ligand factor or Steel factor. Nucleic acid sequences encoding stem cell factors are described in WO92/00376, e.g. the xcex9428 MGF stem cell factor.
Preferably, the vector is a retroviral vector such as MFG or the pBabe vector series. Alternatively, present invention could employ a lentiviral vector producer cell line. In the viral display aspect of the invention, as it is known that the envelope glycoproteins of lentiviral vectors can be substituted by the envelope proteins of C-type retroviruses, the chimeric envelope glycoproteins described below could be used with lentiviral vectors such as those based on HIV, CAEV or Visna. Further vectors suitable for use in the methods described herein can be readily identified by the skilled person.
Typically, the desired protein or polypeptide will be one that a patient is unable to synthesise in his or her body or does not synthesise in the usual amount. An example of this is the use of gene therapy to treat adenosine deaminase dependent severe combined immunodeficiency (ADA-SCID). However, the concepts described herein are applicable to situations in which the nucleic acid encodes a protein or polypeptide that binds a substance that is overexpressed in a patient""s body, e.g. causing some harmful physiological effect, or a protein or polypeptide that can bind to a polypeptide that is produced in a patient""s body in an inactive form to activate it or in an active form to inactivate it. Preferably, the use of the present invention in these applications has the advantage that the therapy provided by transfecting the stem cells is long lasting or permanent, thereby helping to avoid the need for frequently repeated treatment.
Diseases that might be treated using the methods and materials described herein include all forms of chronic granulomatous disease (CGD), all forms of severe combined immunodeficiency (SCID), hyper gamma globulinaemia syndrome (hyper IgM), Wiskott-Aldrich Disease (WAS), thallassaemia, sickle-cell anaemia, other anaemias due to deficiencies of red blood cell proteins, neutrophil defects due to failure to synthesise granule components, e.g. myeloperoxidase deficiency, haemophilia and other clotting disorders such as complement deficiencies, lysomal storage disorders, such as Gaucher""s disease, Hurler""s disease, and mucopolysaccharidosis, leukocyte adhesion deficiency (LAD), bare lymphocyte syndrome, cancer and AIDS.
Other applications of the invention include the genetic modification of haematopoietic stem cells to repopulate the immune system with genetically modified T-lymphocytes that resist HIV, the genetic modification of haematopoeitic stem cells to repopulate bone marrow with haematopoietic progenitors that resist the myelosuppressive effects of cytotoxic chemotherapy, and the genetic modification of T-lymphocytes with chimeric T-cell receptors to target cytotoxic T-cells against tumours or virally infected cells.
In a further aspect, the present invention provides compositions comprising a retroviral packaging cell line or retroviral particles set out above, in combination with a suitable carrier. In this aspect, the present invention provides pharmaceutical compositions suitable for delivering nucleic acid encoding a desired polypeptide to a population of stem cells in vitro, e.g. to prepare engineered stem cells for subsequent implant into a patient. Alternatively, the composition could be used in vivo, to directly deliver the nucleic acid to a patient""s own stem cells. In this case, the composition preferably comprises a retroviral vector incorporating the nucleic acid encoding a desired protein or polypeptide and displaying a growth factor on its surface, e.g. as part of an envelope protein.
In a further aspect, the present invention provides the use of a retroviral cell line or retroviral particles as described above in the preparation of a medicament for treating a disease or disorder that responds to the polypeptide encoded by the nucleic acid packaged in the retroviral particles.
In this aspect, preferably the medicament comprising the retroviral packaging cell line or retroviral particles is administered by implantation into a patient""s bone marrow or is administered by infusion into a patient""s blood. In order to allow the packaging cells to target the bone marrow when administered by infusion, advantageously, receptors such as integrins can be expressed on the surface of the cells. Alternatively or additionally, the immunogenicity of the packaging cells can be reduced by expressing an immunosuppressive factor such as FAS-ligand on the cell surface which can bind to activated T-cell FAS-receptors, triggering the T-cells to die by apoptosis. FAS-ligand expressing allogeneic cell implants have previously been shown to resist immune mediated rejection.
In a further aspect, the present invention provides a method of transforming a population of quiescent cells with nucleic acid encoding a polypeptide so that the nucleic acid is incorporated into the genome of the cells, the method comprising exposing the cells to a retroviral packaging cell line or retroviral particles as described above, wherein the surface bound growth factor induces the cells to divide, so that the nucleic acid encoding the polypeptide for treating a disease or disorder contained in the viral particles can incorporate into the genome of the cells.
In this aspect, preferably the quiescent cells are a population of bone marrow cells enriched in haematopoeitic stem cells.
In further aspects, the present invention provides a population of cells produced by the above method having the nucleic acid encoding a polypeptide for treating a disease or disorder stably incorporated into their genome, and pharmaceutical compositions comprising these populations of cells.
In a further aspect, the present invention provides a method for introducing nucleic acid encoding a polypeptide for treating a disease or disorder into the genome of a population of cells in viva, the method comprising administering a retroviral packaging cell line or retroviral particles by implantation into a patient""s bone marrow or by infusion into a patient""s blood.
By way of example, the present invention will now be described in more detail with reference to the accompanying figures. The following examples are provided to illustrate the present invention, and should not be interpreted as limiting the scope of the claims.